Project Summary Hematopoietic stem cells (HSCs) are the foundation of the adult hematopoietic system. These self-renewing progenitors that reside in the adult bone marrow generate all mature blood lineages through life. Clinically, HSCs are the therapeutic component of bone marrow transplants, used in the treatment of both malignant and non-malignant blood disorders. Inconsistent availability of donors, limitations in graft material, and the possible use of in vitro derived HSCs as a platform for gene therapy and gene editing approaches to treatment of disease has heightened interest in directed differentiation of HSCs from pluripotent precursors, such as induced pluripotent stem (iPS) cells. But despite decades of research, generation of true HSCs with high efficiency engraftment and full multilineage potential remains impossible, suggesting that key specification signals remain to be determined. An obvious means of instructing HSC specification in vitro is by attempting to recapitulate the normal embryonic inductive processes. Across vertebrate phyla, HSCs are specified from developing arterial endothelium, most notably in the primitive descending aorta of mammalian embryos, or its cognate, the dorsal aorta in anamniotic vertebrates such as zebrafish. Significant progress towards generation of the immediate precursor to HSCs, the ?hemogenic endothelium?, has been made in recent years, but the full set of inductive signals that work directly on this endothelium to initiate the definitive hematopoietic program has not been determined. Specification signals likely come from neighboring cells that form an inductive specification ?niche.? As nothing is known about the origin or composition of such cells, we set out to define cell types that might contribute to the specification niche. Hematopoietic programming is highly conserved from mammals to zebrafish, and in vivo observation of tissue specification and hematopoietic development is highly accessible in fish, due to their rapid development, optical transparency at the times when HSCs are specified, and the availability of diverse transgenic animals in which tissue and cell types are labeled by fluorescence transgenes. In preliminary studies with zebrafish, we have determined that neural-crest-derived cells are the first known cell type contributing to the HSC specification niche. Our results show that neural crest cells physically contact hemogenic endothelium shortly before the HSC program initiates. Multiple separate perturbations causing defects in neural crest specification or morphogenesis lead to a loss of HSCs, demonstrating that proper neural crest patterning is required for HSC specification. These data establish neural crest cells as a key component of the inductive HSC specification niche, indicating that signals they present are critical to instruct initiation of the hematopoietic program. Here we propose to identify the unknown HSC specification signals presented by neural crest, and establish the conservation of neural crest contribution to the HSC specification niche in mammals.